Evaluating the function of murine quiescent hematopoietic stem cells following non-homologous end joining-based genome editing

Kohei Shiroshita; Hiroshi Kobayashi; Keiyo Takubo

doi:10.1016/j.xpro.2023.102347

Evaluating the function of murine quiescent hematopoietic stem cells following non-homologous end joining-based genome editing

STAR Protoc. 2023 Jun 9;4(2):102347. doi: 10.1016/j.xpro.2023.102347. Online ahead of print.

Authors

Kohei Shiroshita¹, Hiroshi Kobayashi¹, Keiyo Takubo²

Affiliations

¹ Department of Stem Cell Biology, Research Institute, National Center for Global Health and Medicine, Tokyo 162-8685, Japan.
² Department of Stem Cell Biology, Research Institute, National Center for Global Health and Medicine, Tokyo 162-8685, Japan. Electronic address: keiyot@gmail.com.

Abstract

Preculture is indispensable for achieving highly efficient non-homologous end joining (NHEJ)-based genome editing. Here, we present a protocol for optimizing genome editing conditions for murine hematopoietic stem cells (HSCs) and evaluating their function following NHEJ-based genome editing. We describe steps for sgRNA preparation, cell sorting, preculture, and electroporation. We then detail post-editing culture and transplanting of bone marrow. This protocol can be used to study genes related to HSC quiescence. For complete details on the use and execution of this protocol, please refer to Shiroshita et al.¹.

Keywords: CRISPR; Stem Cells.